PROJECT SUMMARY In chronic hepatitis B virus (HBV) infection, the viral genome forms a stable minichromosome, the covalently closed circular DNA (cccDNA), which can persist throughout the lifespan of the hepatocyte. Targeting the clearance of the viral minichromosome may result in a cure of chronic HBV infection. Current antiviral therapy (ART) targets the virus reverse transcriptase, and rarely establishes immunological control over HBV replication driven by cccDNA. HBV-specific CD8+ cytotoxic T lymphocytes (CTLs) can mediate the killing of infected hepatocytes and accelerate the clearance of cccDNA. However, HBV-specific CTLs are deleted, dysfunctional or succumb to exhaustion in patients with chronic HBV infection. Adoptive cell transfer (ACT) of HBV-specific CTLs is a highly promising treatment for chronic HBV infection. Naive or central memory T cell- derived effector CTLs known as ?highly reactive? cells are optimal populations for cell-based therapies, because these cells have a high proliferative potential, are less prone to apoptosis than terminally differentiated cells and have the higher ability to respond to homeostatic cytokines. However, such ACT is often not feasible due to difficulties in obtaining sufficient numbers of CTLs from patients. The long-term goal of our research program is to develop and optimize strategies for utilizing pluripotent stem cells (PSCs) as a source of highly reactive CTLs for cell-based therapies. The objective in this application is to study the potential elimination of HBV cccDNA reservoirs by immune-mediated cell killing using highly reactive HBV- specific CTLs from PSCs (i.e., PSC-CTLs). The central hypothesis is that HBV-specific PSC-CTLs developed by our system of differentiation are naive single-type viral Ag-specific CD8+ T cells, which can accumulate in HBV-infected liver and mediate the killing of infected hepatocytes after adoptive transfer and act as excellent candidates for cell-based therapies in chronic HBV infection. Guided by published results and preliminary data from the applicant's laboratory, this hypothesis will be tested by pursuing two specific aims: 1) to define the fundamental properties of HBV-specific PSC-CTLs that relate to cytotoxicity; and 2) to study cell-based therapies of chronic HBV infection using viral Ag-specific PSC-CTLs. Under the first aim, in vitro and in vivo approaches, which have been established as feasible in the applicant's laboratory, will be used. The cell properties of HBV-specific PSC-CTLs regarding phenotype, specificity and function will be defined. Under the second aim, using in vitro and in vivo models of HBV replication, cell-based therapies for chronic HBV infection using viral Ag-specific iPSC-CTLs will be optimized. The proposed research is innovative, because the concept of targeting HBV cccDNA reservoirs using viral Ag-specific PSC-CTLs has not been previously explored. The proposed research is significant, because it will provide new insights into therapeutic use of stem cell-derived T lymphocytes. HBV combination therapies using standard ART and ACT of viral Ag-specific PSC-CTLs likely results in a cure of chronic HBV infection.